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(Circulation. 2000;102:3015.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Endotoxin-Induced Mortality Is Related to Increased Oxidative Stress and End-Organ Dysfunction, Not Refractory Hypotension, in Heme Oxygenase-1–Deficient Mice

Philippe Wiesel, MD; Anand P. Patel, MS; Nicole DiFonzo, BS; Pooja B. Marria, BS; Chäng U. Sim, BS; Andrea Pellacani, MD, PhD; Koji Maemura, MD, PhD; Brian W. LeBlanc, BS; Kathryn Marino, BS; Claire M. Doerschuk, MD; Shaw-Fang Yet, PhD; Mu-En Lee, MD, PhD; Mark A. Perrella, MD

From the Program of Developmental Cardiovascular Biology, the Cardiovascular Division (P.W., A.P.P., A.P., K. Maemura., S.-F.Y., M.-E.L., M.A.P.) and the Pulmonary and Critical Care Division (M.A.P.), Brigham and Women’s Hospital; the Department of Medicine (A.P., S.-F.Y., M.-E.L., M.A.P.) Harvard Medical School; and the Cardiovascular Biology Laboratory (P.W., A.P.P., N.D., P.B.M., C.U.S., A.P., K. Maemura, S.-F.Y., M.-E.L., M.A.P.) and the Physiology Program (B.W.L., K. Marino, C.M.D.), Harvard School of Public Health, Boston, Mass. Dr Doerschuk is now at the Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio.

Correspondence to Mark A. Perrella, MD, Program of Developmental Cardiovascular Biology, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail mperrella{at}rics.bwh.harvard.edu

	Abstract

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Background—Heme oxygenase (HO)-1 is an enzyme that degrades heme to generate CO (a vasodilatory gas), iron, and the potent antioxidant bilirubin. A disease process characterized by decreases in vascular tone and increases in oxidative stress is endotoxic shock. Moreover, HO-1 is markedly induced in multiple organs after the administration of endotoxin (lipopolysaccharide [LPS]) to mice.

Methods and Results—To determine the role of HO-1 in endotoxemia, we administered LPS to mice that were wild-type (+/+), heterozygous (±), or homozygous null (-/-) for targeted disruption of HO-1. LPS produced a similar induction of HO-1 mRNA and protein in HO-1^+/+ and HO-1^+/- mice, whereas HO-1^-/- mice showed no HO-1 expression. Four hours after LPS, systolic blood pressure (SBP) decreased in all the groups. However, SBP was significantly higher in HO-1^-/- mice (121±5 mm Hg) after 24 hours, compared with HO-1^+/+ (96±7 mm Hg) and HO-1^+/- (89±13 mm Hg) mice. A sustained increase in endothelin-1 contributed to this SBP response. Even though SBP was higher, mortality was increased in HO-1^-/- mice, and they exhibited hepatic and renal dysfunction that was not present in HO-1^+/+ and HO-1^+/- mice. The end-organ damage and death in HO-1^-/- mice was related to increased oxidative stress.

Conclusions—These data suggest that the increased mortality during endotoxemia in HO-1^-/- mice is related to increased oxidative stress and end-organ (renal and hepatic) damage, not to refractory hypotension.

Key Words: endotoxin • shock • vasoconstriction • perfusion

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Sepsis is a disease process caused by a severe underlying infection.^{1 2} If left unchecked, this process may progress to refractory hypotension, multiple organ system failure, and death. Beyond refractory hypotension, oxygen-derived free radicals are believed to contribute to the cellular and tissue injury associated with endotoxin-induced inflammation. Investigators have suggested that this oxidative damage may be a major cause of organ failure and mortality associated with sepsis³ and that the administration of antioxidants may be an adjuvant to conventional therapy (such as vasoconstrictors) in the management of sepsis.⁴ Other than the cardiovascular response and its associated hypotension, abnormalities of the renal, hepatic, pulmonary, and hematologic systems are common during endotoxemia. In the course of sepsis, the development of multiple organ failure and of pulmonary nosocomial infection⁵ contributes significantly to morbidity and mortality.

Heme oxygenase (HO)-1 is a stress response enzyme that is induced by stimuli associated with oxidative stress.⁶ HO-1 catalyzes the degradation of heme to generate biliverdin, CO, and iron.⁷ Biliverdin is subsequently converted to bilirubin, a potent endogenous antioxidant.⁸ CO shares many similarities with NO, such as its ability to increase cGMP levels and promote vasodilation, thereby modulating tissue perfusion.^{9 10} We have previously demonstrated that interleukin-1ß and lipopolysaccharide (LPS) markedly induce HO-1 expression in cultured vascular smooth muscle cells and several organs of endotoxemic rats, respectively,^{11 12} suggesting that HO-1 may be involved in the pathogenesis of endotoxic shock. These data are supported by human studies showing an elevation in carboxyhemoglobin levels in septic trauma patients.¹³ We have also demonstrated that Zn-protoporphyrin IX, an inhibitor of HO activity, abrogates endotoxin-induced hypotension in rats.¹¹ These results imply that the marked induction of HO-1 during endotoxemia contributes to the decrease in systemic blood pressure. Conversely, other investigators have shown that the administration of LPS to rats receiving high doses of HO inhibitors¹⁴ or mice lacking HO-1¹⁵ leads to increased mortality. Using high doses of LPS (25 mg/kg), investigators have shown that the increased mortality in HO-1 null (HO-1^-/-) mice is associated with hepatic necrosis in vivo.¹⁵ Taken together, these results suggest that although an exaggerated induction of HO-1 may participate in the hypotensive response to LPS, basal HO-1 expression is needed to resist oxidative stress.

To better understand the role of HO-1 in the pathophysiology of endotoxemia, we evaluated LPS-induced hypotension and end-organ dysfunction in HO-1^-/- mice.¹⁶ The goal of the present study was to define the role of HO-1 in LPS-induced hypotension and to determine whether refractory hypotension and/or exaggerated oxidative stress was responsible for the mortality in HO-1^-/- mice.

	Methods

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Mouse Model of Endotoxemia
Mice that were wild-type (+/+), heterozygous (±), or homozygous null (-/-) for targeted disruption of HO-1¹⁶ were studied. These mice were maintained on a 129SvxBALB/c genetic background, and littermates were used for the studies. Salmonella typhosa LPS (Sigma Chemical Co) was administered intraperitoneally (5 mg/kg) to 20- to 30-g female mice that were 16 weeks of age. Pilot experiments revealed that 5 mg/kg LPS produced a 15% to 20% decrease in systolic blood pressure (SBP). Mice were injected with 0.9% saline to act as controls for animals receiving LPS. HO-1^-/- mice receiving LPS were also treated with the endothelin (ET) type A and B (ET_A/ET_B) receptor antagonist PD142893 (3 µmol/kg IP,¹⁷ Sigma) or the antioxidant N-acetylcysteine (NAC, 150 mg/kg). The mice were killed 24 hours after LPS administration. Tissue and plasma samples were collected and stored at -80°C until further processing. The Harvard Medical Area Standing Committee on Animals approved the protocol.

Northern Blot Analysis
Total RNA was obtained from mouse tissue by guanidinium isothiocyanate extraction and silica-gel-membrane spin technology (RNeasy midi kit, Qiagen). Northern blot analysis was performed as described,^{11 12} and filters were hybridized with ³²P-labeled rat HO-1, rat HO-2, or mouse ET-1 probes. To correct for differences in RNA loading, the filters were also hybridized with a ³²P-labeled oligonucleotide probe complementary to 28S ribosomal RNA. Images were displayed, and radioactivity was measured on a PhosphorImager running ImageQuant software (Molecular Dynamics).

Western Blot Analysis
Tissue samples were homogenized in 13.2 mmol/L Tris-HCl, 5.5% glycerol, 0.44% SDS, and 10% ß-mercaptoethanol. An equal amount of soluble protein (50 µg) was fractionated by Tris-glycine-SDS–polyacrylamide gel (12%) electrophoresis, and Western blotting was performed as described¹² with use of an antibody to recombinant rat HO-1 or HO-2 protein (1:1000, Stressgen).

Blood Pressure Measurements
Baseline SBP was measured 12 hours before the administration of LPS or saline vehicle and then 4 and 24 hours after LPS or saline vehicle. A tail-cuff method was used to measure SBP. Mice were trained by placing them in restraints, 1 hour daily, for 10 days before the experiments. Once they were fully trained, conscious mice were restrained and gently warmed with use of a heating lamp. An occlusion cuff and a piezoelectric pulse sensor were placed around the tail (Kent Scientific), and SBP was measured after a 15-minute acclimatization period. A minimum of 8 serial measurements were made, and the average value was calculated (Mac Laboratory software, version 3.5, AD Instruments). Both training and blood pressure measurements were performed the same time each day (afternoon).

Biochemical Measurements
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Cr) levels were measured in plasma by use of commercial kits (Sigma), according to the manufacturer’s recommendations. Lipid peroxidation products were measured in liver tissue by use of the lipid peroxidation assay kit from Calbiochem. This colorimetric assay is specific for malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE). Values are expressed as the sum of MDA+4-HNE levels (micromoles MDA+4-HNE per gram wet tissue).

Characterization of Lung Neutrophils, Edema, and Alveolar Destruction
Twenty-four hours after intratracheal administration of LPS (5 mg/kg), the percentage of distal lung tissue occupied by neutrophils, edema, or destroyed alveolar walls was quantified on paraffin-embedded histological sections by using point-counting techniques.^{18 19} Total circulating white blood cell (WBC) and neutrophil counts were measured with use of a hemocytometer and blood smears stained with Leukostat (Fisher Scientific).

Statistics
Where indicated, comparisons between groups were made by factorial ANOVA followed by the Fisher least significant difference test when appropriate. Comparisons of mortality between groups were made by the ² goodness of fit test. Statistical significance was accepted at P<0.05. Data are expressed as mean±SE.

	Results

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LPS Induction of HO-1 Expression in Multiple Organs
We initially evaluated HO-1 expression in the livers, kidneys, and lungs of HO-1 wild-type mice at baseline and then 4 and 24 hours after LPS administration. HO-1 mRNA was markedly induced by LPS in all organs after 4 and 24 hours (Figure 1). When comparing HO-1 expression 24 hours after LPS, we found that HO-1 mRNA was markedly induced in both HO-1^+/+ and HO-1^+/- mice (Figure 2a). As expected, HO-1 mRNA was not detected in HO-1^-/- mice. A constitutive isoform of HO (HO-2) was not affected by LPS in these organs. Moreover, HO-2 was not upregulated in HO-1^-/- mice compared with HO-1^+/+ and HO-1^+/- mice (in the presence or absence of LPS); thus, HO-2 did not compensate for the absence of HO-1 (Figure 2a). To determine whether these changes in HO-1 mRNA levels translated into similar changes in HO-1 protein levels, we subjected liver protein extracts to Western blot analysis. Again, the induction of HO-1 was similar in HO-1^+/+ and HO-1^+/- mice, whereas no HO-1 protein was detected in the HO-1^-/- mice (Figure 2b). HO-2 protein levels were not increased by LPS, and they were not upregulated in HO-1^-/- mice compared with HO-1^+/+ and HO-1^+/- mice (Figure 2b).

View larger version (20K): [in this window] [in a new window]   Figure 1. LPS induction of HO-1 mRNA in liver, kidney, and lung tissue in vivo. HO-1+/+ mice were injected with vehicle (-) or LPS (5 mg/kg) intraperitoneally. Mice were killed 4 or 24 hours after LPS administration, and total RNA was extracted from liver, kidney, and lung tissue. Northern blot analysis was performed as described (Methods), and nitrocellulose filters were hybridized with 32P-labeled rat HO-1 probe. Filters were also hybridized with 32P-labeled oligonucleotide probe complementary to 28S ribosomal RNA to control for differences in loading. Signal intensity of each RNA sample hybridized to HO-1 probe was divided by that of each sample hybridized to 28S probe. Normalized signal intensities were then plotted as percentage of vehicle (mean±SE). Groups of mice were as follows: vehicle (-), n=3; 4 hours, n=3; and 24 hours, n=6.

View larger version (32K): [in this window] [in a new window]   Figure 2. Comparable induction of HO-1 expression by LPS in HO-1+/+ and HO-1+/- mice and no expression in HO-1-/- mice. a, HO-1+/+, HO-1+/-, and HO-1-/- mice were injected intraperitoneally with vehicle (C) or LPS (5 mg/kg). They were killed 24 hours after LPS administration, and total RNA was extracted from liver, kidney, and lung tissue. Northern blot analysis was performed as described (Methods), and nitrocellulose filters were hybridized with 32P-labeled rat HO-1 and HO-2 probes and 32P-labeled oligonucleotide probe complementary to 28S ribosomal RNA to assess for differences in loading. b, Liver tissue from HO-1+/+, HO-1+/-, and HO-1-/- mice injected with vehicle or LPS (as described in panel a) was homogenized, and Western blot analysis was performed as described (Methods). HO-1 and HO-2 proteins were detected by immunoblotting with polyclonal rabbit antibodies against recombinant rat HO-1 and HO-2. In panels a and b, data are representative of at least 2 experiments.

Recovery of LPS-Induced Hypotension in HO-1^-/- but Not HO-1^+/+ and HO-1^+/- Mice
HO-1–derived CO is a vasoactive gas that may participate in the regulation of vascular tone. Therefore, we measured SBP in HO-1^+/+, HO-1^+/-, and HO-1^-/- mice challenged with LPS. SBP was not different between the groups at baseline. Four hours after LPS, the decrease in SBP was similar in all the groups; however, after 24 hours, SBP was significantly higher in HO-1^-/- mice (121±5 mm Hg) compared with HO-1^+/+ (96±7 mm Hg) and HO-1^+/- (89±13 mm Hg) mice (Figure 3). These data suggest that HO-1 contributes to the sustained, but not the acute, hypotension associated with endotoxemia.

View larger version (19K): [in this window] [in a new window]   Figure 3. Resolution of LPS-induced hypotension in HO-1-/- mice. Baseline SBP was measured in HO-1+/+ (solid circles, n=10), HO-1+/- (solid triangles, n=8), and HO-1-/- (open circles, n=8) mice as described (Methods). Mice were injected with LPS (5 mg/kg IP), and SBP was subsequently measured 4 and 24 hours after LPS. *P<0.05 vs HO-1+/+ and HO-1+/- groups.

Twenty-four hours after LPS administration, we assessed mortality in the HO-1^+/+, HO-1^+/-, and HO-1^-/- mice. Despite the lack of sustained hypotension in HO-1^-/- mice, mortality was increased (60%, P<0.05) compared with HO-1^+/+ (10%) and HO-1^+/- (10%) mice.

Sustained Induction of ET-1 mRNA in HO-1^-/- Mice After LPS Administration
In addition to its direct vasodilatory effect, CO is also known to inhibit the expression of ET-1, a potent vasoconstrictor.²⁰ Thus, we hypothesized that in HO-1^-/- mice, unabated ET-1 gene expression may contribute to the restoration of blood pressure. We performed Northern blot analysis to evaluate ET-1 mRNA levels in the kidney, an organ that robustly expresses ET-1 and plays a pivotal role in the regulation of blood pressure.²¹ Four hours after LPS administration, the induction of ET-1 mRNA was similar in HO-1^+/+ and HO-1^-/- mice (Figure 4a). In HO-1^+/+ mice, ET-1 mRNA had returned to baseline levels by 24 hours. However, at this time point, ET-1 mRNA levels were still markedly increased in HO-1^-/- mice (Figure 4a). Twenty-four hours after LPS, the increase in ET-1 mRNA was also noted in liver and lung tissue from HO-1^-/- mice (Figure 4b). These data suggest that the lack of HO-1 allowed a sustained induction of ET-1 message in multiple organs.

View larger version (21K): [in this window] [in a new window]   Figure 4. Sustained induction of ET-1 mRNA in absence of HO-1. a, HO-1+/+ (solid bars) and HO-1-/- (open bars) mice were injected with vehicle (-) or LPS (5 mg/kg) intraperitoneally. Mice were killed 4 or 24 hours after LPS administration, and total RNA was extracted from kidneys. Each experiment was performed 4 times. b, HO-1+/+ (solid bars) and HO-1-/- (open bars) mice were injected with LPS (5 mg/kg) intraperitoneally and were killed 24 hours later; total RNA was extracted from livers (n=4), kidneys (n=6), and lungs (n=3). In panels a and b, Northern blot analysis was performed as described (Methods), and nitrocellulose filters were hybridized with 32P-labeled mouse ET-1 probe and 32P-labeled oligonucleotide probe complementary to 28S ribosomal RNA to control for differences in loading. Signal intensities were normalized and plotted as described in Figure 1. c, HO-1-/- mice received the ETA/ETB receptor antagonist PD142893 (3 µmol/kg IP, n=3, open circle) or vehicle (n=6, solid circle) 4 hours before and 20 hours after LPS administration (5 mg/kg IP). SBP was subsequently measured 24 hours after LPS. *P<0.05 vs vehicle group.

To determine whether this sustained induction of ET-1 contributed to the higher SBP in HO-1^-/- mice after 24 hours of LPS, we administered an ET_A/ET_B receptor antagonist (PD142893, 3 µmol/kg IP) or vehicle to HO-1^-/- mice. Before LPS administration, the ET_A/ET_B receptor antagonist caused no change in SBP (data not shown). However, in the presence of LPS, HO-1^-/- mice receiving the ET_A/ET_B receptor antagonist had a significantly lower SBP (90±7 mm Hg) compared with SBP in null mice receiving vehicle (121±5 mm Hg) (Figure 4c). SBP in HO-1^-/- mice receiving the ET_A/ET_B receptor antagonist was comparable to SBP in HO-1^+/+ and HO-1^+/- mice after 24 hours of LPS (Figure 3).

End-Organ Dysfunction in Endotoxemic HO-1^-/- Mice
LPS at a dose of 5 mg/kg caused no hepatocellular destruction by histological analysis; however, iron deposition was present in the liver (data not shown). To further determine whether end-organ damage occurred in HO-1^-/- mice in the absence of prolonged hypotension, we evaluated markers of hepatic and renal insult 24 hours after LPS administration. Plasma levels of AST and ALT were measured to evaluate the occurrence of hepatocellular injury. Levels of both transaminases were similar in HO-1^+/+, HO-1^+/-, and HO-1^-/- mice at baseline (Figure 5a). However, AST and ALT levels were markedly induced in HO-1^-/- mice after LPS but not in HO-1^+/+ and HO-1^+/- mice. Similarly, there was no difference in plasma Cr levels between the groups at baseline (Figure 5b). After LPS administration, there was a significant increase in plasma Cr in HO-1^-/-mice but not in HO-1^+/+ and HO-1^+/- mice. These data show a significant LPS-induced deterioration in hepatic and renal function in the absence of HO-1.

View larger version (17K): [in this window] [in a new window]   Figure 5. LPS–induced liver and renal dysfunction in absence of HO-1. HO-1+/+ (solid bars), HO-1+/- (gray bars), and HO-1-/- (open bars) mice were injected with vehicle (C, n=3 in each group) or LPS (5 mg/kg, n=3 in each group) intraperitoneally. Mice were killed 24 hours after LPS administration, and plasma levels for ALT and AST (a) and Cr (b) were measured. *P<0.05 vs all other groups.

Increased Susceptibility to LPS-Induced Oxidative Damage in the Absence of HO-1
Oxidative stress induces HO-1 in a variety of cell types and pathophysiological conditions, and HO-1 constitutes an important antioxidant defense mechanism. To determine whether the absence of HO-1 may lead to increased oxidative damage in target organs of endotoxemic mice, we measured 2 aldehydes (MDA and 4-HNE) that are generated during lipid peroxidation (Figure 6a). A specific colorimetric method was used to detect both aldehydes (see Methods) from liver homogenates. MDA+4-HNE levels were similar at baseline in HO-1^+/+ and HO-1^-/- mice (Figure 6a). Twenty-four hours after LPS administration, a 3-fold increase in aldehyde levels was evident in HO-1^+/+ mice. However, MDA+4-HNE levels were induced >7-fold in HO-1^-/- mice receiving LPS. These data demonstrate increased oxidative liver injury in the absence of HO-1.

View larger version (13K): [in this window] [in a new window]   Figure 6. Lipid peroxidation products are increased in absence of HO-1, and antioxidants can decrease death rate of HO-1-/- mice receiving LPS. a, HO-1+/+ (solid bars) and HO-1-/- (open bars) mice were injected with vehicle (-, n=3 in each group) or LPS (+, 5 mg/kg, n=3 in each group) intraperitoneally. Mice were killed 24 hours after LPS administration, and lipid peroxidation products (MDA and 4-HNE) were measured in liver homogenates. *P<0.05 vs vehicle-treated HO-1+/+ group; P<0.05 vs all other groups. b, HO-1-/- mice received the antioxidant NAC (150 mg/kg IP, n=6) or vehicle (n=10) 1 hour before and 12 hours after LPS administration (5 mg/kg IP). Death rate was assessed after 24 hours. *P<0.05 vs no (-) NAC treatment.

To determine whether increased oxidative stress and damage may have contributed to increased organ dysfunction and mortality, HO-1^-/- mice receiving LPS were treated with vehicle or the antioxidant NAC (150 mg/kg). Mortality was assessed after 24 hours. In contrast to vehicle-treated mice that had a death rate of 60%, mice receiving NAC had a death rate of 0% after receiving LPS (Figure 6b). Moreover, end-organ dysfunction improved in the presence of NAC. Plasma Cr was similar to baseline in HO-1^-/- mice receiving NAC before LPS (26±4 µmol/L); however, plasma Cr was significantly increased in the absence (56±2 µmol/L) of NAC.

No Increased Lung Damage in HO-1^-/- Mice Exposed to LPS but Increased Circulating Inflammatory Cells
Similar to the histological analysis of the liver, there were no abnormalities in the lung tissue of HO-1^-/- mice after intraperitoneal administration of LPS (data not shown). In addition, there was no gross increase in lung iron deposition in these mice. Because pulmonary nosocomial infections and acute lung injury are associated with increased morbidity and mortality in septic patients, the inflammatory response during LPS-induced pneumonia was evaluated in HO-1^-/- and HO-1^+/+ mice.^{18 19} There was no difference in distal airway neutrophil accumulation, pulmonary edema, or alveolar destruction in HO-1^-/- mice compared with HO-1^+/+ mice (Figure 7a). Baseline numbers of circulating WBCs were not different between the groups. However, WBCs in HO-1^-/- mice increased after LPS administration compared with WBCs in HO-1^+/+ mice (Figure 7b). We next examined the circulating level of neutrophils, a critical source of inflammatory mediators during endotoxemia.²² There was a marked increase in circulating neutrophils in HO-1^-/- mice, but not in HO-1^+/+ mice, after LPS administration (Figure 7b).

View larger version (18K): [in this window] [in a new window]   Figure 7. No increased structural damage in lung to LPS administration but increased circulating inflammatory cells in absence of HO-1. a, HO-1+/+ (solid bars, n=6) and HO-1-/- (open bars, n=6) mice were injected with LPS (5 mg/kg intratracheally). Percentages of distal lung tissue occupied by neutrophils, edema, and destroyed alveolar walls were quantified after 24 hours by use of point-counting techniques as described (Methods). NS indicates not significant. b, HO-1+/+ (solid bars, n=3) and HO-1-/- (open bars, n=7) mice were injected with LPS (5 mg/kg intratracheally), and circulating WBCs and neutrophils were quantified after 24 hours. *P<0.05 for HO-1-/- group vs HO-1+/+ group.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Sepsis is a systemic response to a severe underlying infection. Even with intense efforts to improve therapeutic strategies, mortality rates remain high, and the incidence of sepsis continues to rise.²³ In some patients, intractable hypotension leads to death, whereas in others, the failure of one or more organs is the cause of demise. Beyond antibiotics, supportive care remains the mainstay of therapy at present.^{24 25} If the blood pressure remains low, vasoconstrictor agents are administered. Although these agents increase blood pressure, they also have the potential to decrease end-organ perfusion and promote organ damage.²⁴ Thus, a delicate balance exists between vasoconstrictors and vasodilators during septic shock. Systemic blood pressure needs to be increased to reverse hypotension, yet this cannot occur at the expense of peripheral organ perfusion.

In the present study, SBP was measured before and after (4 and 24 hours) the administration of LPS to HO-1^+/+, HO-1^+/-, and HO-1^-/- mice. Four hours after LPS, SBP was reduced in mice from all 3 groups. However, SBP was significantly increased after 24 hours in HO-1^-/- mice compared with HO-1^+/+ and HO-1^+/- mice (Figure 3). SBP was not different between HO-1^+/+ and HO-1^+/- mice, and HO-1 expression was similar (Figure 2). Even though SBP was higher in HO-1^-/- mice after LPS, we found increased mortality. These data demonstrate for the first time that HO-1 contributes to the sustained hypotension associated with endotoxemia and that a lack of HO-1 results in increased mortality that is not associated with intractable hypotension.

An endogenous vasoconstrictor that plays an important role in the pathophysiology of septic shock is ET-1.²⁶ Investigators have suggested that the release of endogenous ET-1 during endotoxemia may help to counteract severe hypotension,²⁷ especially in the setting of NO synthase inhibition.²⁸ However, the increased levels of ET-1 may lead to excessive vasoconstriction in peripheral vascular beds, and ET-1 contributes to the dysfunction of multiple organs during endotoxemia, including liver,²⁹ kidney,³⁰ and lung.³⁰ Because HO-1–derived CO is known to suppress ET-1,²⁰ we investigated whether ET-1 mRNA levels would be altered in HO-1^-/- mice. ET-1 was induced by LPS in the kidneys of HO-1^+/+ and HO-1^-/- mice after 4 hours; however, ET-1 mRNA levels remained elevated only in HO-1^-/- mice after 24 hours (Figure 4a). The sustained increase in ET-1 mRNA was also noted in the liver and lungs of HO-1^-/- mice (Figure 4b). Administration of an ET_A/ET_B receptor antagonist to HO-1^-/- mice receiving LPS resulted in a significantly lower SBP compared with SBP in HO-1^-/- mice receiving vehicle (Figure 4c). Moreover, none of the null mice receiving the ET_A/ET_B receptor antagonist died after 24 hours of LPS stimulation (data not shown). These data suggest that increased levels of ET-1 in multiple organs contributed not only to higher SBP but also to increased mortality in HO-1^-/- mice receiving LPS.

The development of multiple organ failure is a key predictor of outcome in septic patients.³¹ Thus, we examined the mice for LPS-induced end-organ damage. HO-1^-/- mice at this age showed no evidence of increased organ dysfunction (Figures 5a and 5b), oxidative damage (Figure 6a), or systemic inflammation (Figure 7b) compared with HO-1^+/+ mice at baseline. However, after LPS stimulation, plasma ALT and AST levels dramatically increased in HO-1^-/- mice (Figure 5a) in the absence of intractable hypotension. Furthermore, plasma Cr levels were significantly increased in HO-1^-/- mice after LPS administration (Figure 5b). The end-organ insults were not present in HO-1^+/+ or HO-1^+/- mice exposed to LPS. These data show LPS-induced hepatocellular damage and renal dysfunction only in mice lacking HO-1.

The absence of HO-1–induced CO and the sustained induction of ET-1 may have contributed to end-organ damage due to oxidative damage, resulting from decreased tissue perfusion. Furthermore, decreased generation of bilirubin (an important antioxidant⁸ ) and iron deposition provide an environment susceptible to oxidative stress and damage in the absence of HO-1. The administration of LPS to wild-type mice caused a 3-fold increase in lipid peroxidation products in liver tissue (MDA+4-HNE, Figure 6a), which is consistent with increased oxidative stress associated with endotoxemia. In HO-1^-/- mice, however, lipid peroxidation products were increased 7-fold after the administration of LPS. This level of lipid peroxidation products in HO-1^-/- mice was significantly higher than that in wild-type mice (Figure 6a). Previously, investigators have shown that HO-1–deficient cells from mice¹⁵ and humans³² in culture are more sensitive to injury by pro-oxidants (such as hemin, hydrogen peroxide, paraquat, and cadmium chloride). In the present study, we show that the administration of a pathophysiological stimulus, LPS, can enhance tissue oxidative damage in HO-1^-/- mice in vivo. The importance of increased oxidative stress and damage in these mice was underscored by experiments demonstrating that the administration of an antioxidant, NAC, improved end-organ dysfunction and prevented LPS-induced death in HO-1^-/- mice (Figure 6b).

Studies have suggested that HO helps to protect the lung from oxidant-induced injury. For example, Dennery et al³³ showed that mice lacking HO-2 were more susceptible to hyperoxia-induced injury, whereas Otterbein et al³⁴ demonstrated that overexpression of HO-1 can protect the lung from hyperoxia. This latter effect of HO-1 appears to be mediated, in part, by the attenuation by CO of neutrophil infiltration into the airways.³⁵ In patients with sepsis, nosocomial infections and acute lung injury are complications associated with increased morbidity and mortality.⁵ Thus, we evaluated LPS-induced pneumonia¹⁸ in HO-1^-/- mice to determine whether the lack of HO-1 would enhance lung injury. Unexpectedly, in this model, we found no increase in distal airway neutrophil accumulation, pulmonary edema, or alveolar destruction in HO-1^-/- mice compared with HO-1^+/+ mice (Figure 7a). However, circulating WBCs were increased in HO-1^-/-, but not HO-1^+/+, mice exposed to LPS (Figure 7b). This increase was due primarily to a marked increase in circulating neutrophils (a critical source of inflammatory mediators) in HO-1^-/- mice. These data suggest that the release of neutrophils from the bone marrow may be enhanced. This response may represent a globally exaggerated systemic inflammatory response.

Taken together, we show that endotoxin-induced mortality in mice lacking HO-1 is not the result of intractable hypotension but is associated with end-organ damage (liver and renal) and increased oxidative stress. These data suggest that in conjunction with supportive care and the use of vasoconstrictor agents to treat hypotension, strategies aimed at reducing inflammation and oxidative injury in target organs during sepsis may have therapeutic benefit.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-03194 and HL-60788 (to Dr Perrella), GM-53249 (to Dr Lee), and HL-48160 (to Dr Doerschuk); an American Heart Association Grant-in-Aid (to Dr Perrella); grants from Novartis and the SICPA Foundation (to Dr Wiesel); and a grant from the Bristol-Myers Squibb Pharmaceutical Research Institute. We dedicate this study to the memory of Dr Mu-En Lee, who was a constant source of inspiration and support for our work. We also thank Dorothy Zhang for her technical assistance.

	Footnotes

 
Drs Wiesel and Patel contributed equally to this study.

Received April 24, 2000; revision received June 20, 2000; accepted June 30, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

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